The long term goal of our studies is to understand control of gene expression in situ within the dynamic environment of the bone cell nucleus. During the past funding period, using the Runx transcription factors as a paradigm, we identified and initiated characterization of the first intranuclear trafficking signal that directs these osteogenic and hematopoietic regulatory proteins to punctate, nuclear matrix associated sites that support gene expression. This project will now combine molecular, biochemical, cellular, and in vivo genetic approaches to focus on establishing the developmental function and mechanisms by which Runx factors are targeted to distinct subnuclear foci where the regulatory machinery for transcription is recruited, assembled and organized. The findings from these studies will provide the in situ context for the integration of osteogenic signals (Project 2) into the molecular instructions required for controlling the chromatin packaging of bone phenotypic genes to establish competency for protein/DNA and protein/protein interactions that mediate activity (Project 3). The central hypothesis of our project is that the targeting of Runx2 to specific subnuclear foci is required for biological control of skeletal gene transcription. The specific aims are directed to establishing: (i) the, physiological requirement of Runx 2 subnuclear targeting for normal skeletal development and bone formation in vivo; (ii) the role of Runx2 intranuclear trafficking in osteoblast differentiation and bone specific gene transcription; (iii) the dynamics of targeting Runx2 and its co regulators to subnuclear foci in live cells in response to skeletal regulatory stimuli; and (iv) the molecular mechanisms that regulate intranuclear trafficking to Runx2 foci to support bone specific transcriptional control.